![]() ![]() ( C ) IL1beta concentrations determined in GIT1 fl/fl and GIT1 CKO mice treated with clodronate liposomes (CLOD) or control liposomes (CON) (one-way ANOVA with post hoc test). ( A, B ) Concentration of IL1beta ( A ) and TNFalpha ( B ) present in the bone defect region at five time points (days 0, 3, 7, 10, and 14) after injury from GIT1 fl/fl and GIT1 CKO mice were measured by ELISA (one-way ANOVA with post hoc test). Enhanced IL1beta production and higher proportion of M1-like macrophages infiltrated in bone defects of GIT1 CKO mice on days 3 and 7 post-injury. adoptive transfer of NC-BMDMs under macrophage depletion (NC-įig. macrophage depletion (DT) P < 0.01, 0.001 vs. Data represent results of six mice/group. Transfer of NC-BMDMs, but not siPou4f1-BMDMs, restored both a-SMA expression and a-SMA + CD68 + MMT cells. ( B ) Flow cytometry analysis, and ( C ) real-time PCR show that DT-induced macrophage depletion reduced both total a-SMA expression and a-SMA + CD68 + MMT cells. However, transfer of siPou4f1-BMDMs restored only the F4/80 + macrophage population. Adoptive transfer of NC-BMDMs restored all of these populations and collagen I deposition in the IRI model. Compared to the IRI control, DT treatment reduced F4/80 + macrophages, a-SMA + myofibroblasts, Pou4f1 + interstitial cells, and interstitial collagen I deposition. ( A ) Immunohistochemistry staining for Pou4f1 ( Upper ) and Col-I ( Middle ), and confocal microscopy for a-SMA (green) and F4/80 (red) (power panel). At 24 h after IRI surgery, mice were injected with either Pou4f1-knockdown (siPou4f1) or nonsense siRNA treated (NC) BMDM (2 x 10 6 cells/mouse) which had a 24-h stimulation with TGF-b1 (5 ng/mL) in culture prior to injection. Mice were treated with DT for 3 d before IRI surgery to deplete macrophages. A day 7 IRI model was performed in LysM-Cre/DTR mice. Silencing of Pou4f1 in BMDMs inhibits MMT in an adoptive transfer version of the IRI model. Data represent means +- SEM from three experiments. D, E Quantification of association of platelets with liver Kupffer cells or Kupffer cell (KC) number at 2 h after alpha neuraminidase injection. ![]() C Representative immunofluorescent images showing association of platelets (CD41, red) with Kupffer cells (F4/80, blue) in the liver sections. Platelet count was measured using flow cytometric analysis of peripheral blood based on anti-CD41. B Percentage of platelets of either WT or Clec4f -/- recipients treated with Arthrobacter ureafaciens -derived neuraminidase at different time points. A Flow cytometry analysis of galactose exposure (RCA I binding) on platelets of either WT or Clec4f -/- recipients treated with Arthrobacter ureafaciens -derived neuraminidase at different time points. 2 Kupffer cell CLEC4F is a dominant receptor in mediating the rapid clearance of desialylated platelets in the liver. ( F ) IF staining for intrahepatic platelets (CD41 + ) and MFs (F4/80+) was performed 3įig. ( D ) Serum levels of ALT and ( E ) liver histology were evaluated 24 hr after APAP treatment (n = 6 mice/group). ( D-F ) Chil1 -/- mice reconstituted with rmChi3l1 were treated with either Ctrl IgG or alpha-podoplanin Ab for 16 hr and subsequently challenged with APAP. ( C ) Immunofluorescence (IF) staining of liver sections for podoplanin and F4/80 is shown and the proportions of MFs that express Pdpn were quantified, Scale bar, 25 mum. ( B ) MFs were isolated and mRNA levels of Pdpn in MFs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Chil1 -/- and Cd44 -/- mice were treated with PBS or rmChi3l1 followed by APAP challenge simultaneously and mice were sacrificed 3 hr after APAP (n = 3 mice/group). ( B, C ) Wild-type (WT) mice were treated with APAP. After 3 hr, mice were sacrificed and MFs were isolated to measure mRNA levels of various adhesion molecules, including selectin P ligand ( Selplg ), Cd40, melanoma cell adhesion molecule ( Mcam ), Fc receptor ( Fcr ), intercellular adhesion molecule 1 ( Icam1 ), lymphocyte function-associated antigen 1 ( Lfa1 ), von Willebrand factor ( Vwf ), and podoplanin ( Pdpn ). ( A ) Male WT, Chil1 -/-, Cd44 -/- mice were treated with acetaminophen (APAP) (n = 4 mice/group). Chitinase 3-like-1 (Chi3l1)/CD44 signaling in MFs upregulates podoplanin expression and platelet adhesion.
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